Fig 1: Malat1 binds to Tead3 to inhibit Nfatc1 activity and osteoclastogenesis.a. RNA immunoprecipitation (RIP) assay. Tead proteins were immunoprecipitated from cross-linked RAW264.7 cells by a pan-Tead-specific antibody. Tead-bound Malat1 was quantitated by qPCR. The nuclear RNA U6 was used as a negative control.b. Immunoblotting of Yap, Tead1, Tead2, Tead3,Tead4, and Hsp90 in B16F1, MEF, BMM, MSC, L929, and RAW264.7 cells.c. RNA pulldown assay. Unlabeled and biotinylated (Btn) Malat1 fragments (P1-P6) were synthesized by in vitro transcription (left panel), incubated with the lysate of HEK293T cells overexpressing V5-tagged Tead3, and pulled down with streptavidin beads. The bound proteins were eluted by boiling in Laemmli sample buffer and immunoblotted with a V5-specific antibody (right panel).d. Immunoblotting of Nfatc1, Ctsk, Tead1, Tead2, Tead3, Tead4, and ß-actin in RAW264.7 cells treated with RANKL (50 ng/mL) for the indicated times.e. HEK293T cells co-transfected with SFB-Nfatc1 and MYC-Tead3 were subjected to pulldown with S-protein beads or a MYC-specific antibody, followed by immunoblotting with antibodies against MYC and FLAG.f, g. Control and MALAT1-knockout HEK293T cells co-transfected with SFB-Nfatc1 and MYC-Tead3 were subjected to pulldown with a MYC-specific antibody (f) or S-protein beads (g), followed by immunoblotting with antibodies against MYC and FLAG.h, i. MALAT1-knockout and Malat1-restored HEK293T cells co-transfected with SFB-Nfatc1 and MYC-Tead3 were subjected to pulldown with a MYC-specific antibody (h) or S-protein beads (i), followed by immunoblotting with antibodies against MYC and FLAG.j. Graphical representation of mouse Nfatc1 and Tead3 proteins and their truncation mutants. The upper panel shows full-length (FL) Nfatc1 and five truncation mutants: NT (N-terminal region), Mid (middle region = DNA-binding domain, DBD), CT (C-terminal region), NT + Mid, and Mid + CT. The lower panel shows full-length Tead3 and two truncation mutants: NT (N-terminal region = DNA-binding domain) and CT (C-terminal region = YAP-binding domain).k. HEK293T cells co-transfected with MYC-Tead3 and SFB-tagged full-length or truncated Nfatc1 were subjected to pulldown with S-protein beads, followed by immunoblotting with antibodies against MYC and FLAG.l. HEK293T cells co-transfected with SFB-Nfatc1 and MYC-tagged full-length or truncated Tead3 were subjected to pulldown with a MYC-specific antibody, followed by immunoblotting with antibodies against MYC and FLAG.m, n. HEK293T cells were co-transfected with Tead3, constitutively active Nfatc1 (Nfatc1-CA), a Renilla luciferase reporter, and a firefly luciferase reporter containing tandem Nfatc1-binding sites (m) or the Ctsk promoter (n). Luciferase activity was measured 48 hours after transfection.o, p. Wild-type, MALAT1-knockout, and Malat1-restored HEK293T cells were co-transfected with increasing amounts of Tead3, Nfatc1-CA, a Renilla luciferase reporter, and a firefly luciferase reporter containing tandem Nfatc1-binding sites (o) or the Ctsk promoter (p). Luciferase activity was measured 48 hours after transfection.q, r. Control and Malat1-knockdown RAW264.7 cells were transfected with Tead3 siRNA or scrambled negative control (NC). 24 hours after siRNA transfection, the cells were treated with RANKL for 5 days, followed by TRAP staining and quantification (q) of multinucleated TRAP-positive cells (outlined by dashed lines). Scale bars, 100 µm. Cell lysates were subjected to immunoblotting of Nfatc1, Ctsk, and ß-actin (r).s. Model for the regulation of osteoclastogenesis by the Malat1–Tead3–Nfatc1 axis.Statistical significance in a and m-q was determined by a two-tailed unpaired t-test. Error bars are s.e.m.